Ch 21 Principles of Chromatography and Mass Spectrometry Ch 22 Gas and Liquid Chromatography What is Chromatography? Sec 21-1 Chromatography = a process where compounds in a mixture are separated by passing it through a column that retains some compounds longer than others Mobile phase = Stationary phase = Elution = Different Types of Chromatography 1.Adsorption chromatography stationary phase = solid (analyte adsorbs onto) mobile phase = gas or liquid 2.Partition chromatography stationary phase = thin liquid film coating inside surface of column or coats the solid support (SiO 2 ) mobile phase = gas or liquid 3.Ion-exchange chromatography stationary phase = charged resin with covalently bound ionic groups such as -SO 3 - or -N(CH 3 ) 3+ ; electrostatically attracts ionic analytes mobile phase = liquid 4.Molecular exclusion chromatography e.g. gel filtration, gel permeation, molecular sieve smaller molec ules trapped in pores while the larger ones elute faster 6.Affinity chromatography most selective, covalently bonded antibody binds a specific protein How We Describe a Chromatogram Sec 21-2 N = 5.55 t r 2 /W 2 1/2 H = L/N Theoretical Plates Resolution = t r /W av 1. Longitudinal Diffusion (B/u) Why Do Bands Spread? Sec 21-3 2. Equilibration Time (Cu) 3. Eddy Diffusion (A) The Van Deemter Equation Practical Control of Separation Find optimum flow rate (u opt ) Decrease the stationary phase thickness Temperature programming increases D s Choose carrier gas with higher D m Decrease solid support particle size Narrow-bore columns Internal Standards Sec 5-4 Area concentration (Area X) / (Area S) unknown (Area X) / (Area S) standard = [X] / [S] unknown [X] / [S] standard X = unknown S = standard ASK YOURSELF (5-D, p. 103) - Using an Internal Standard A mixture containing 52.4 nM iodoacetone (X) and 38.9 nm p-dichlorobenzene (S) gave the relative detector response (area of X)/(area of S) = A solution containing an unknown quantity of X plus 742 nM S gave a relative detector response (area of X)/(area of S) = Find the concentration of X in the unknown. Gas Chromatography Sec 22-1 Sample volatilized and injected into a column along with an inert carrier gas or MOBILE PHASE Mixture separated by differential retention by the STATIONARY PHASE (some molecules hjeld up longer than others) Components separate according to boiling point (lowest first) Match analyte polarity to stationary phase polarity (Like dissolves Like) Basic Instrumentation The Injection Port Capillary Columns Stationary Phases Temperature Programming Detectors A. Thermal Conductivity B. Flame Ionization Detector (FID) C. Electron Capture Detector (ECD) Applications of Gas Chromatography Routine separation (e.g. after a synthesis) Identification of an unknown by comparing retention time to a known standard Quantization using an INTERNAL STANDARD Example Separations A. Environmental EPA Methods B. Biological